The core idea of this proposal is that: (a) substances released by neurons trigger the activation of glia, & (b) this glial activation, in turn, leads to the release of glial products that induce & maintain pain facilitation. It is clear from prior studies that spinal cord glia (microglia and astrocytes) become activated in response to signaling by small diameter sensory afferents. It is also clear from prior studies that blocking either glial activation or the action of glial products prevents & reverses diverse enhanced pain states. What is not understood is the signal(s) released by neurons which cause(s) such glial activation. The overall goal of this proposal is to understand this key step. We have recently discovered one putative neuron-to-glia signal so will focus on it. This signal is fractalkine, a protein tethered to the exterior surface of spinal cord neurons & sensory afferents. When neurons become strongly activated, fractalkine can break free to form a diffusible signal. We have shown in spinal cord that (a) microglia (& possibly astrocytes) express receptors for fractalkine; (b) injecting fractalkine induces thermal hyperalgesia & mechanical allodynia; & (c) early evidence suggests that fractalkine may potentially be an important signal for initiating & maintaining neuropathic pain. We propose to use a multidisciplinary approach to examine fractalkine-induced neuron-to-glia communication. First, we will define how critical fractalkine is for the induction & long-term maintenance of neuropathic pain by (a) testing the effect of a selective fractalkine receptor antagonist delivered prior to, or up to 3 months after, nerve damage & (b) testing knockouts for both fractalkine and its receptor (CX3CR1). Second, we will define the effects of fractalkine on microglia versus astrocytes using a combination of approaches: pharmacological, immunohistochemical, in vitro & adoptive transfer (intrathecal injection of in vitro stimulated microglia & astrocytes). Last, as time permits, we will examine whether fractalkine creates pain facilitation via proinflammatory cytokine release &/or "priming" of gila. Together, this series of studies will provide new insights into how fractalkine in particular, & neuron-to-gila signaling in general, creates & maintains pain facilitation. Understanding this key step between neural activation & glial activation may have implications for developing new approaches for pain control.